The CAP released this month a new book titled An Algorithmic Approach To Hemostasis Testing. On this and the following pages is the chapter on normal PT and aPTT by Timothy Hayes, MD, DVM, of the Department of Pathology, Maine Medical Center, Portland. But first, a few words from the book's editor, Kandice Kottke–Marchant, MD, PhD, chair of the Pathology and Laboratory Medicine Institute and section head of hemostasis and thrombosis, Cleveland Clinic, and chair of the Department of Pathology, Cleveland Clinic Lerner College of Medicine.
Laboratory testing for bleeding and thrombotic disorders often requires the performance and interpretation of many assays because of the complexity of the hemostatic system, which involves numerous coagulation proteins, platelets, von Willebrand factor, the fibrinolytic system, and the vasculature. Furthermore, appropriately collecting, processing, and storing blood samples for hemostasis testing offers more challenges than any other area in the laboratory. An Algorithmic Approach To Hemostasis Testing is a new textbook that offers a practical and comprehensive guide for pathologists and laboratories engaged in hemostasis testing and will serve as a test-interpretation resource for pathologists and clinicians alike.
This textbook grew out of many publications by members of the CAP Coagulation Resource Committee that were directed toward best practices for hemostasis testing and use of algorithms to direct testing and patient diagnosis. This book was originally conceived as a compilation of those publications, but the committee instead produced a cohesive and completely rewritten text. It includes sections on hemostasis physiology, the basics of hemostasis laboratory testing and interpretation, an algorithmic approach to bleeding disorders and venous thrombophilic disorders, and monitoring anticoagulant drugs, together with illustrative patient case studies. Sample collection and processing as well as patient history and diagnostic criteria also are covered.
—Kandice Kottke-Marchant, MD, PhD
Timothy E. Hayes, MD, DVM
When confronted with an acutely bleeding patient, the most pressing question posed by the clinician is often, “What do I do to make the bleeding stop?” This question is particularly important because the clinician can select from multiple therapeutic options, including procedures (cauterization, surgical intervention, etc.), medications (desmopressin acetate [DDAVP], antifibrinolytics, etc.), and transfusion of blood components (platelets, frozen plasma, cryoprecipitate, and specific clotting factors, such as concentrates of factor VIII, factor IX, von Willebrand factor [vWF], and now even recombinant factor VIIa). The most appropriate clinical response depends upon accurate identification of the underlying cause for the bleeding. The prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are often used in the initial evaluation of a bleeding patient to specifically assess the potential role of defective secondary hemostasis as an underlying cause of the bleeding. Not infrequently, both test results are within normal limits. This chapter will discuss an approach to this clinical setting.
Defects in secondary hemostasis are common causes for increased bleeding risk. Examples include liver disease, vitamin K deficiency, dilutional coagulopathies, disseminated intravascular coagulation (DIC), inherited factor deficiencies, and medications, such as heparins, warfarins, and the direct thrombin inhibitors (DTIs). The PT and aPTT are frequently used to screen for these types of defects and, in general, are well suited for such use. In particular, these assays are widely available, easily and rapidly performed, relatively inexpensive, and fairly sensitive for the more common defects in secondary hemostasis. It is not uncommon for these two assays to be within normal limits in a patient who is reported to be actively bleeding or to have a personal or family history of a bleeding tendency.
Before undertaking an exhaustive and expensive laboratory evaluation for a hemorrhagic disorder, it is good practice to have some communication between the laboratory and the patient’s clinician. Good communication and an adequate understanding of clinical and laboratory hemostasis are instrumental in making appropriate decisions about the next steps, both in terms of ensuring a good patient outcome as well as appropriate use of medical resources.
As a starting point, it is not unreasonable to assess the significance of the bleeding in question. In some cases, the bleeding is determined to be within normal limits (for example, a certain amount of chest tube drainage is not unusual after some forms of cardiac surgery) and therefore may not warrant an extensive laboratory evaluation. An experienced clinician is in the best position to determine the “normal” amount of bleeding for the clinical setting. At the same time, it may be worthwhile discussing the likelihood that the bleeding is due to something other than a coagulopathy (e.g., inadequate surgical hemostasis).
If the bleeding is thought to be clinically significant and an underlying coagulopathy cannot be excluded, then further laboratory evaluation is indicated. The patient’s clinical information, especially the personal/family history and current medications, are important in determining the type and extent of the testing to be performed. [Chapter five of the book contains additional information on this subject.]
False-negative (normal) PT and aPTT
Although the PT and aPTT are fairly sensitive for detecting most of the common defects in secondary hemostasis, they have their limitations. Causes of false-negative results may be broadly divided into four categories: patient specific, preanalytic, analytic, and postanalytic [see algorithm, at right].
Clinically significant defects in secondary hemostasis may not be detectable by the PT and aPTT in several settings. Therapeutic interventions, such as transfusion of plasma-containing blood components (including cryoprecipitate, prothrombin complex concentrates, factor concentrates, etc.) and administration of medications (including vitamin K, DDAVP, etc.) may transiently correct the underlying coagulation defect. Pregnancy and the use of oral contraceptives (ethinyl estradiol, mestranol) may result in elevations of several procoagulant factors, including factors VII, VIII, XII, von Willebrand factor (vWF), and fibrinogen. Factor XI may decrease or increase during pregnancy. An acute phase process resulting from inflammatory and stressful states (including strenuous exercise, high altitude, and mental stress) may be associated with elevated levels of fibrinogen, vWF, and factor VIII. Laboratory specimens drawn in these clinical settings may yield normal test results.
There are numerous variables in the preanalytic phase of testing that may result in a falsely normal (false-negative) PT and/or aPTT. Erroneous results obviously may arise from drawing blood from the wrong patient. The phlebotomy process may also introduce errors. A specimen obtained via a traumatic or repeated venipuncture effort may introduce tissue juice (thromboplastin) contamination, which may shorten the aPTT, especially if there is also a slow flow of blood into the tube. Shortening of the PT and/or aPTT may also be seen with excessive mixing of the specimen, hemolysis, and chilling of the specimen. All of these events may bring an otherwise abnormal (prolonged) PT/aPTT result down into the normal range (false-negative result). When in doubt, the PT and aPTT should be repeated on a freshly collected specimen. [Chapter four of the book contains additional information on this subject.]
The sensitivity of the PT and aPTT to detect clinically significant factor deficiencies varies with the reagent used. For example, the sensitivity of the PT reagent is highly dependent upon the source of thromboplastin, while the phospholipid composition and the type of activator influence the sensitivity of the aPTT reagent. The sensitivity of aPTT reagents to factor deficiencies varies not only between manufacturers, but also between lots of reagents from the same manufacturer. These concerns may be somewhat mitigated by understanding these reagent-specific issues and carefully establishing the assay reference range. Some of the clinically significant mild hemophilias and von Willebrand disease may have an aPTT result that is just a few seconds above the upper limit of the reference range. A poorly generated reference range therefore may not discern these patients from normal. [Chapter six of the book contains additional information on this subject.]
Postanalytic errors might include transcription errors, calculation errors, and oral miscommunication of test results. For example, it is critical that the correct international sensitivity index (ISI), geometric mean of the reference range, and calculations are used in generating the international normalized ratio (INR). This is becoming more important as many laboratories have completely replaced PT reporting with the INR—even for non-warfarin patients.
If it is determined that the patient with a normal PT and aPTT has a compelling bleeding history that may be due to a coagulopathy, and the above issues have been addressed, then it may be of value to consider additional testing, as outlined below.
Primary hemostasis defects
Defects in primary hemostasis, such as von Willebrand disease and platelet dysfunction, are a common cause of bleeding and should therefore be considered during the evaluation of a hemorrhagic disorder. Although von Willebrand disease typically presents with an elevated aPTT, patients with some subtypes of von Willebrand disease present with bleeding despite a normal PT and aPTT. [Chapters 14 and 16 of the book contain additional information on this subject.]
Secondary hemostasis defects
The aPTT is used to detect coagulation abnormalities of the intrinsic pathway. It is more sensitive to deficiencies of the proximal part of the pathway (factors XII, XI, IX, and VIII) than to the distal part (factor II and fibrinogen). Therefore, it is not unreasonable to include a fibrinogen level (factor I) in the initial evaluation of a bleeding patient with a normal PT and aPTT.
The hereditary hemophilias include deficiencies of factors VIII (hemophilia A), IX (hemophilia B), and XI (hemophilia C). Most aPTT reagents will yield prolonged (abnormal) test results when factor VIII or IX drop below 30% to 40% of normal, but some reagents have failed to detect mild hemophilia levels as low as 25% (e.g., a normal aPTT). Most patients with heterozygous factor XI deficiency will be asymptomatic, yet some will bleed, especially after trauma. These patients may have a normal aPTT.
The family history may be helpful but may also be misleading. For example, the lack of a family history for a bleeding disorder does not exclude a hereditary factor deficiency. As many as one-third of cases of hemophilia A and B may be due to recent mutations—most commonly being first detected in the carrier female—with no previous family bleeding history. Therefore, specific factor assays should be performed when there is a strong clinical suspicion of mild hemophilia, even if the aPTT is normal.
Factor XIII deficiency
Factor XIII (FXIII) is a cysteine enzyme (transglutaminase) that is activated by thrombin. The active enzyme covalently cross-links fibrin molecules to each other and cross-links α2-antiplasmin to fibrin. This cross-linking converts the loose fibrin polymer into a highly organized structure with increased tensile strength that imparts clot stability and resistance to fibrinolysis. FXIII also participates in wound healing, cell migration, and clot retraction.
FXIII deficiency may be either inherited or acquired. Bleeding is usually not significant unless FXIII deficiency is severe (levels below 3%). The inherited form is an autosomal recessive disorder and is highly heterogeneous from a molecular perspective. It is relatively rare, with an estimated frequency of 1 in 2,000,000. Bleeding is variable, but usually severe. Umbilical site bleeding within a few days of birth occurs in roughly 80% of cases. Delayed bleeding after mild trauma is typical. Intracranial bleeding may be spontaneous or occur after mild trauma. Subcutaneous and muscle hematomas are common. Delayed bleeding and menorrhagia are seen. Hemarthrosis is uncommon. Pregnancies may be lost due to placental bleeding. Poor wound healing occurs in about 14% of patients. Some patients present with a milder form of bleeding. It has been reported that heterozygotes may also have bleeding symptoms.
An acquired deficiency may rarely be seen with FXIII inhibitors, which are typically IgG antibodies. These antibodies may arise after exposure to certain drugs, including penicillin, phenytoin, valproate, and isoniazid. These antibodies may be associated with a severe deficiency of FXIII and significant bleeding. Acquired deficiency of FXIII may also be associated with certain malignancies (leukemia, plasmacytoma), sepsis, and DIC. The FXIII deficiency associated with these clinical conditions is usually mild and of minimal clinical significance.
The routine clotting assays (PT, aPTT, thrombin time, and fibrinogen) are normal even in severe FXIII deficiency. The qualitative screening assay is the clot solubility test. Patient plasma is incubated with thrombin to form a clot. This clot is then suspended in a 5M urea solution or 1% monochloroacetic acid. With severe FXIII deficiency (level <3% of normal), normal fibrin cross-linking has not stabilized the fibrin clot and it dissolves within 60 minutes. An abnormal screening test result should be followed with mixing studies with normal plasma to rule out an inhibitor and further confirmed by a quantitative assay (functional and/or immunologic assays).
Defects in fibrinolysis may be associated with significant bleeding and a normal PT and aPTT. Hereditary forms are rare. Acquired forms are more common and are typically associated with thrombolytic therapy or DIC. [Chapter 13 of the book contains additional information on this subject.]
Certain vascular disorders, such as vasculitis, connective tissue disorders, vascular malformations, senile purpura, and steroid-induced purpura, may be associated with a bleeding tendency and a normal PT and aPTT. A variety of conditions can be association with a bruising tendency, including Ehlers-Danlos syndrome, hereditary hemorrhagic telangiectasia, osteogenesis imperfecta, Cushing syndrome, vitamin C deficiency (scurvy), Marfan syndrome, Fabry disease, and others. A complete discussion of these vascular disorders is beyond the scope of this book; however, the coagulation laboratory’s role in the diagnosis of these disorders may include testing to exclude platelet disorders, DIC, or immune thrombocytopenic purpura (ITP), which may present as microvascular hemorrhage. Other pathology laboratories may be involved in skin biopsy and studies to exclude cutaneous vasculitis, such as testing for complement, antinuclear antibodies and antineutrophilic cytoplasmic antibody (ANCA).
An algorithmic approach to evaluating the setting of bleeding with a normal PT and aPTT is shown. This algorithm is just one of many possible strategies that might be used.
Approach to the bleeding patient
Clark P, Brennand J, Conkie JA, McCall F, Greer IA, Walker ID. Activated protein C sensitivity, protein C, protein S and coagulation in normal pregnancy. Thromb Haemost. 1998;79:1166–1170.
Dzik WH. Component therapy before bedside procedures. In: Mintz PD, ed. Transfusion Therapy: Clinical Principles and Practice. 2nd ed. Bethesda, Md: AABB Press; 2005:1–26.
Goodnight SH, Hathaway WE. Screening tests in hemostasis. In: Goodnight SH, Hathaway WE. Disorders of Hemostasis and Thrombosis: A Clinical Guide. 2nd ed. New York: McGraw-Hill; 2001:41–51.
Konkle BA. Clinical approach to the bleeding patient. In: Colman RW, Clowes AW, Godhaber SZ, Marder VJ, George JN, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 5th ed. Philadelphia, Pa: Lippincott Williams and Wilkins; 2006:1147–1158.
Schafer AI. Approach to bleeding. In: Loscalzo J, Schafer AI, eds. Thrombosis and Hemorrhage. 3rd ed. Philadelphia, Pa: Lippincott Williams and Wilkins; 2003: 315–329.
Factor XIII deficiency
Anwar R, Miloszewski KJA. Factor XIII deficiency. Br J Haematol. 1999;107:468– 484.
Dargaud Y, de Mazancourt P, Rugeri L, et al. An unusual clinical presentation of factor XIII deficiency and issues relating to the monitoring of factor XIII replacement therapy. Blood Coagul Fibrinolysis. 2008; 19(5):447–452.
Francis JL. The detection and measurement of factor XIII activity: a review. Med Lab Sci. 1980;37:137–147.
Greenberg CS, Sane DC, Lai TS. Factor XIII and fibrin stabilization. In: Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, George JN, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 5th ed. Philadelphia, Pa: Lippincott Williams and Wilkins; 2006:317–334.
Seitz R, Duckert F, Lopaciuk S, et al. ETRO Working Party on factor XIII questionnaire on congenital factor XIII deficiency in Europe: status and perspectives. Semin Thromb Hemost. 1996;22:415–418.
Baxter BT. Heritable diseases of the blood vessels. Cardiovasc Pathol. 2004;14: 185– 188.
Myllyharju J, Kivirikko KI. Collagens and collagen-related diseases. Ann Med. 2001;33:4–6.
Zumberg M, Kitchens CS. Purpura and other hematovascular disorders. In: Kitchens CS, Alving BM, Kessler CM, eds. Consultative Hemostasis and Thrombosis. 2nd ed. Philadelphia, Pa: Saunders; 2007: 159–182.